Effect of the structural isomer N-3-fluorenylacetamide on microsomal binding and hydroxylation of the carcinogen N-2-fluorenylacetamide

N-2-Fluorenylacetamide (2-FAA), a carcinogen, or N-3-fluorenylacetamide (3-FAA), the noncarcinogenic isomer, when added to hepatic microsomes from 3-methylcholanthrene (3-MC)-treated rats, in phosphate buffer-l,2-propanediol, exhibited equal affinity for cytochrome P₁-450 as indicated by equal binding constants (K_s). Both isomers were bound to the same site on cytochrome P₁-450 and displaced each other from the common binding site, as shown by competitive inhibition. Microsomal hydroxylation of 2-FAA to N-OH-2-FAA and to 7-OH-2-FAA was also inhibited by 3-FAA. The inhibitory effect was enhanced by omission of 1,2-propanediol from the incubation system. In contrast to the inhibition of the binding of 2-FAA by 3-FAA, the inhibition of hydroxylation of 2-FAA by 3-FAA was noncompetitive or uncompetitive. The contrasting patterns of inhibition of hydroxylation of 2-FAA by 3-FAA and of binding of 2-FAA by the isomer indicated that the binding site of 2-FAA is separate from the site of hydroxylation. This conclusion was supported by (1) a 10³-fold difference in the values of K_m, and K_m of 2-FAA, and (2) opposite effects of 1,2-propanediol on binding and hydroxylation of 2-FAA. Whereas binding of 2-FAA to cytochrome P₁-450 was increased with increasing concentrations of 1,2-propanediol, the microsomal formation of N-OH-2-FAA and of 7-OH-2-FAA was markedly diminished under the same conditions.